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Group IIA secreted phospholipase A 2 (group IIA sPLA 2 ) is known to display potent Gram-positive bactericidal activity in vitro and in vivo. We have analyzed the bactericidal activity of the full set of native stingray and dromedary groups V, IIA, and IB sPLA 2 s on several Gram-positive and Gram-negative strains. The rank order potency among both marine and mammal sPLA 2 s against Gram-positive bacteria is group IIA > V > IB, whereas Gram-negative bacteria exhibited a much higher resistance. There is a synergic action of the sPLA 2 with lysozyme when added to the bacteria culture prior to sPLA 2 .The bactericidal efficiency of groups V and IIA sPLA 2 s was shown to be dependent upon the presence of calcium ions and to a less extent Mg 2+ ions and then a correlation could be made to its hydrolytic activity of membrane phospholipids. Importantly, we showed that stingray and dromedary groups V, IIA, and IB sPLA 2 s present no cytotoxicity after their incubation with MDA-MB-231cells. stingray groups V and IIA sPLA 2 s, like mammal ones, may be considered as future therapeutic agents against bacterial infections.

Background Mammalian GIB-PLA2 are well characterized. In contrast, much less is known about aquatic ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes. The aim of this study was to check some biochemical and structural properties of a marine stingray phospholipase A2 (SPLA2). Results The effect of some proteolytic enzymes on SPLA2 was checked. Chymotrypsin and trypsin were able to hydrolyze SPLA2 in different ways. In both cases, only N-terminal fragments were accumulated during the hydrolysis, whereas no C-terminal fragment was obtained in either case. Tryptic and chymotryptic attack generated 13 kDa and 12 kDa forms of SPLA2, respectively. Interestingly, the SPLA2 13 kDa form was inactive, whereas the SPLA2 12 kDa form conserved almost its full phospholipase activity. In the absence of bile slats both native and 12kDa SPLA2 failed to catalyse the hydrolysis of PC emulsion. When bile salts were pre-incubated with the substrate, the native kinetic protein remained linear for more than 25 min, whereas the 12 kDa form activity was found to decrease rapidly. Furthermore, The SPLA2 activity was dependent on Ca 2+ ; other cations (Mg 2+ , Mn 2+ , Cd 2+ and Zn 2+ ) reduced the enzymatic activity notably, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca 2+ . Conclusions Although marine and mammal pancreatic PLA2 share a high amino acid sequence homology, polyclonal antibodies directed against SPLA2 failed to recognize mammal PLA2 like the dromedary pancreatic one. Further investigations are needed to identify key residues involved in substrate recognition responsible for biochemical differences between the 2 classes of phospholipases.

Glycyrrhizin (GL) has been used in Japan to treat patients with chronic viral hepatitis, as an anti-inflammatory drug to reduce serum alanine aminotransferase levels. GL is also known to exhibit various biological activities, including anti-viral effects, but the anti-hepatitis C virus (HCV) effect of GL remains to be clarified. In this study, we demonstrated that GL treatment of HCV-infected Huh7 cells caused a reduction of infectious HCV production using cell culture-produced HCV (HCVcc). To determine the target step in the HCV lifecycle of GL, we used HCV pseudoparticles (HCVpp), replicon, and HCVcc systems. Significant suppressions of viral entry and replication steps were not observed. Interestingly, extracellular infectivity was decreased, and intracellular infectivity was increased. By immunofluorescence and electron microscopic analysis of GL treated cells, HCV core antigens and electron-dense particles had accumulated on endoplasmic reticulum attached to lipid droplet (LD), respectively, which is thought to act as platforms for HCV assembly. Furthermore, the amount of HCV core antigen in LD fraction increased. Taken together, these results suggest that GL inhibits release of infectious HCV particles. GL is known to have an inhibitory effect on phospholipase A2 (PLA2). We found that group 1B PLA2 (PLA2G1B) inhibitor also decreased HCV release, suggesting that suppression of virus release by GL treatment may be due to its inhibitory effect on PLA2G1B. Finally, we demonstrated that combination treatment with GL augmented IFN-induced reduction of virus in the HCVcc system. GL is identified as a novel anti-HCV agent that targets infectious virus particle release.

The endogenous lipid agent N-arachidonoylethanolamine (anandamide), among other effects, has been shown to be involved in nociceptive processing both in the central and peripheral nervous systems. Anandamide is thought to be synthesised by several enzymatic pathways both in a Ca(2+)-sensitive and Ca(2+)-insensitive manner, and rat primary sensory neurons produce anandamide. Here, we show for the first time, that cultured rat primary sensory neurons express at least four of the five known Ca(2+)-insensitive enzymes implicated in the synthesis of anandamide, and that application of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-arachidonoyl, the common substrate of the anandamide-synthesising pathways, results in anandamide production which is not changed by the removal of extracellular Ca(2+). We also show that anandamide, which has been synthesised in primary sensory neurons following the application of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-arachidonoyl induces a transient receptor potential vanilloid type 1 ion channel-mediated excitatory effect that is not inhibited by concomitant activation of the cannabinoid type 1 receptor. Finally, we show that sub-populations of transient receptor potential vanilloid type 1 ion channel-expressing primary sensory neurons also express some of the putative Ca(2+)-insensitive anandamide-synthesising enzymes. Together, these findings indicate that anandamide synthesised by primary sensory neuron via a Ca(2+)-insensitive manner has an excitatory rather than an inhibitory role in primary sensory neurons and that excitation is mediated predominantly through autocrine signalling. Regulation of the activity of the Ca(2+)-insensitive anandamide-synthesising enzymes in these neurons may be capable of regulating the activity of these cells, with potential relevance to controlling nociceptive processing.

Background Mammalian sPLA2-IB are well characterized. In contrast, much less is known about aquatic ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes. Results A marine stingray phospholipase A 2 (SPLA2) was purified from delipidated pancreas. Purified SPLA2, which is not glycosylated protein, was found to be monomeric protein with a molecular mass of 14 kDa. A specific activity of 750 U/mg for purified SPLA2 was measured at optimal conditions (pH 8.5 and 40 °C) in the presence of 4 mM NaTDC and 8 mM CaCl 2 using PC as substrate. The sequence of the first twenty first amino-acid residues at the N-terminal extremity of SPLA2 was determined and shows a close similarity with known mammal and bird pancreatic secreted phospholipases A2. SPLA2 stability in the presence of organic solvents, as well as in acidic and alkaline pH and at high temperature makes it a good candidate for its application in food industry. Conclusions SPLA2 has several advantageous features for industrial applications. Stability of SPLA2 in the presence of organic solvents, and its tolerance to high temperatures, basic and acidic pH, makes it a good candidate for application in food industry to treat phospholipid-rich industrial effluents, or to synthesize useful chemical compounds.